视黄酸通路的启示——从心脏发育到心肌谱系定向分化

多潜能干细胞具有无限增殖的能力,并能够分化为心肌细胞,因此在心脏再生方面拥有巨大潜力.胚胎发育过程为干细胞定向分化提供了重要线索,在过去的几年中,通过操控心脏发育关键通路,在心肌定向分化方面取得了重要进展,但是现有的分化方法仍不能稳定地诱导心肌细胞,表明现有的通路不能有效解决这些问题.视黄酸(RA)通路在心脏发育过程中发挥重要作用,RA缺失会导致心房变小、心室小梁减少、心肌壁增厚且细胞间连接松散.在体外心肌定向分化过程中,RA多用于促进多潜能干细胞向心房分化.但从RA通路基因敲除小鼠的表型来看,除了调控心肌亚型分化,RA在多个发育阶段发挥重要作用.深入解析RA在心肌分化各阶段的作用机制,将有助于获得高质量的心肌细胞.同时,研究RA在心内膜和心外膜分化中的作用机制也有助于解释RA通路敲除小鼠的心脏异常.总之,从RA在胚胎发育中的作用来看,需要更多的体外研究来揭示RA在心肌谱系分化中的作用.本文综述了RA通路在心脏发育的心肌分化过程中的作用,并探讨了需要解决的问题.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.     

Dhrs3 Protein Attenuates Retinoic Acid Signaling and Is Required for Early Embryonic Patterning

All-trans-retinoic acid (atRA) is an important morphogen involved in many developmental processes, including neural differentiation, body axis formation, and organogenesis. During early embryonic development, atRA is synthesized from all-trans-retinal (atRAL) in an irreversible reaction mainly catalyzed by retinal dehydrogenase 2 (aldh1a2), whereas atRAL is converted from all-trans-retinol via reversible oxidation by retinol dehydrogenases, members of the short-chain dehydrogenase/reductase family. atRA is degraded by cytochrome P450, family 26 (cyp26). We have previously identified a short-chain dehydrogenase/reductase 3 (dhrs3), which showed differential expression patterns in Xenopus embryos. We show here that the expression of dhrs3 was induced by atRA treatment and overexpression of Xenopus nodal related 1 (xnr1) in animal cap assay. Overexpression of dhrs3 enhanced the phenotype of excessive cyp26a1. In embryos overexpressing aldh1a2 or retinol dehydrogenase 10 (rdh10) in the presence of their respective substrates, Dhrs3 counteracted the action of Aldh1a2 or Rdh10, indicating that retinoic acid signaling is attenuated. Knockdown of Dhrs3 by antisense morpholino oligonucleotides resulted in a phenotype of shortened anteroposterior axis, reduced head structure, and perturbed somitogenesis, which were also found in embryos treated with an excess of atRA. Examination of the expression of brachyury, not, goosecoid, and papc indicated that convergent extension movement was defective in Dhrs3 morphants. Taken together, these studies suggest that dhrs3 participates in atRA metabolism by reducing atRAL levels and is required for proper anteroposterior axis formation, neuroectoderm patterning, and somitogenesis.     

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-β (Aβ) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aβ-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ∼4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aβ in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy.     

Allopurinol-Enhanced Postischemic Recovery in the Isolated Pat Heart Involves Repletion of High-Energy Phosphates

The effects of allopurinol (AP) on functional and metabolic recovery of the isolated rat heart after global ischemia were studied. Hearts were subjected to aerobic perfusion (30 min), cardioplegic infusion (5 min), normothermic ischemia (37 min), and reperfusion (50 min) which was started with secondary cardioplegic infusion (10 min). AP was injected into rats (44 mg/kg body wt ip 2 h before heart excision) and added to cardioplegic solution (2 mM) prior and after ischemia. AP treatment significantly improved postischemic recovery of the function and reduced the leakage of lactate dehydrogenase from reperfused hearts. These beneficial effects were accompanied by a better preservation of tissue content of ATP, the total adenine nucleotides, phosphocreatine, and the total creatine at the end of reperfusion. Inhibition of xanthine oxidase by AP substantially decreased pre- and postischemic release of xanthine and uric acid and increased postischemic release of hypoxanthine into the coronary effluent. Despite this, AP treated hearts did not exhibit a reduction in hydroxyl radical adduct formation in the effluents at reperfusion assessed by the spin-trap measurements. The results suggest that AP may protect the heart from ischemia/reperfusion injury due to enhanced energy provision rather than by prevention of oxygen-derived free radical formation.     

Retinoic Acid Receptor-Dependent,Cell-Autonomous,Endogenous Retinoic Acid Signaling and Its Target Genes in Mouse Collecting Duct Cells

Background

Vitamin A is necessary for kidney development and has also been linked to regulation of solute and water homeostasis and to protection against kidney stone disease, infection, inflammation, and scarring. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively.

Methodology/Principal Findings

To explore target genes of endogenous tRA/RARs, we employed the mIMCD-3 mouse inner medullary CD cell line, which is a model of CD principal cells and exhibits constitutive tRA/RAR activity as CD principal cells do in vivo. Combining antagonism of RARs, inhibition of tRA synthesis, exposure to exogenous tRA, and gene expression profiling techniques, we have identified 125 genes as candidate targets and validated 20 genes that were highly regulated (Dhrs3, Sprr1a, and Ppbp were the top three). Endogenous tRA/RARs were more important in maintaining, rather than suppressing, constitutive gene expression. Although many identified genes were expressed in UBs and/or CDs, their exact functions in this cell lineage are still poorly defined. Nevertheless, gene ontology analysis suggests that these genes are involved in kidney development, renal functioning, and regulation of tRA signaling.

Conclusions/Significance

A rigorous approach to defining target genes for endogenous tRA/RARs has been established. At the pan-genomic level, genes regulated by endogenous tRA/RARs in a CD cell line have been catalogued for the first time. Such a catalogue will guide further studies on molecular mediators of endogenous tRA/RARs during kidney development and in relation to renal defects associated with vitamin A deficiency.     

维甲酸与精子发生

维生素A的活性代谢物维甲酸在哺乳动物精子发生过程中发挥着重要的调节作用,但其具体调节机制并不十分清楚。该文拟对睾丸内维甲酸的运输、代谢、信号系统以及维甲酸调控精子发生的研究进展进行简单总结。     

Depletion of Retinoic Acid Receptors Initiates a Novel Positive Feedback Mechanism that Promotes Teratogenic Increases in Retinoic Acid

Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA) signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1), a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM) specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.     

Effect of MCI-186 on Postischemic Reperfusion Injury in Isolated Rat Heart

MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a newly developed antioxidant which has been shown to reduce brain edema in cerebral ischemia through inhibition of the lipoxygenase pathway of arachidonic acid. However, its effect on myocardial reperfusion injury after prolonged ischemia has not yet been demonstrated. We compared the mode of the effect of MCI-186 and recombinant human CuZn superoxide dismutase (rh-SOD) in isolated perfused rat hearts subjected to 60-min ischemia followed by 60-min reperfusion. Left ventricular developed pressure (LVDP), necrotic area and the release of creatine phosphokinase (CPK) and endogenous CuZn superoxide dismutase (endoge-SOD) were measured to evaluate myocardial damage. The decrease in left coronary flow (CBF) was measured as an index of the damage of left coronary circulation. MCI-186 (17.5 mg/L) was perfused for 10 min in the MCI group and rh-SOD (70 mg/L) was perfused during the reperfusion period in the SOD group starting 5 min prior to reperfusion. The release patterns of CPK and endoge-SOD were analyzed to elucidate the difference in the mode of protection of MCI-186 and rh-SOD. The LVDP remained higher in both MCI and SOD groups than that of control (76 ± 1, 77 ± 2 and 69 ± 1% of preischemic value, respectively). The necrotic area was significantly attenuated in both MCI and SOD groups compared with that in the control group (16 ± 1,14 ± 1 and 32 ± 170, respectively, p<0.05). Total CPK release was lower in both MCI and SOD groups thfn in the control (78 ± 7, 100 ± 2 and 116 ± 4 × 10³ units/g myocardium respectively). The decrease in CPK release was more marked in the MCI group than that in the SOD group (p<0.05). The reduction in CBF was significantly attenuated by the treatment with rh-SOD or MCI-186, but the effect was much higher in the SOD group than in the MCI group (69 ± 5, 58 ± 2, and 48 ± 2% in SOD, MCI and control groups, respectively). The release pattern of endoge-SOD was identical to that of CPK and thus this did not distinguish the mode of effect of MCI-186 from that of rh-SOD. These results indicate that MCI-186 reduces reperfusion injury in isolated perfused hearts with prolonged ischemia and the effect is more closely related to the reduction of myocyte damage than the preservation of the coronary circulation.